1. The Role of Cysteine String Protein in Synaptic Function
The exocytotic release of neurotransmitter from nerve terminals is a fundamental process underlying most intercellular communication in the nervous system. Synaptic vesicles, the central players in this process, undergo a complex cycle of fusion and fission events. They fuse with the presynaptic membrane in response to a rise in the intracellular Ca2+ concentration, and release their neurotransmitter cargo into the synaptic cleft. The vesicle membrane is then retrieved by endocytosis (Südhof, 1995, Nature 375:645-653; Scales and Scheller, 1999, Nature 401:123-124; Südhof, 2000, Neuron 28:317-320 2000).
In the last ten years, enormous progress has been made in identifying and characterizing the essential protein machinery involved in organizing and maintaining the synaptic vesicle cycle. One component of this complex protein machinery is the cysteine string protein (CSP), which is thought to have an important function in the exocytotic release of neurotransmitter, hormones, and enzyme precursors (Buchner and Gundersen, 1997, Trends Neurosci. 20:223-227). CSP is localized on synaptic vesicles (Mastrogiacomo et al., 1994, Science 263:981-982), chromaffin granules (Chamberlain et al., 1996, J. Biol. Chem. 271:19514-19517), and zymogen granules (Braun and Scheller, 1995, Neuropharmacology 34:1361-1369). It is highly conserved during evolution (Buchner and Gundersen, 1997, Trends Neurosci. 20:223-227), reflected by an overall amino acid identity of almost 55% between rat and Drosophila CSP. Due to a unique structural feature, a string of 11 cysteines flanked on either side by two additional cysteines, it was named cysteine string protein (Zinsmaier et al., 1990, J Neurogenet. 7:15-29). Most of the cysteine residues are palmitoylated and required for membrane targeting of CSP (Gundersen et al., 1994, J. Biol. Chem. 269:19197-19199; Chamberlain and Burgoyne, 1998, Biochem. J. 335:205-209). Another striking feature of CSP is that it contains a J domain at the N terminus. The J domain comprises a stretch of 70 amino acids evolutionarily conserved from E. coli to man (Fink, 1999, Physiol. Rev. 79:425-449).
The biological function of CSP has remained elusive. In Drosophila, deletion of CSP is lethal for a great majority of flies (Zinsmaier et al., 1994, Science 263:977-980). However, a small percentage of CSP null mutants survived and could be analyzed with electrophysiological methods. CSP “knockout” flies showed an impaired presynaptic neuromuscular transmission accompanied with a dramatic loss of synaptic vesicles. It has been suggested that CSP might function in regulating presynaptic Ca2+ channels based on the finding that injection of CSP antisense RNA into Xenopus oocytes inhibited the activity of omega-conotoxin sensitive Ca2+ channels (Gundersen and Umbach, 1992, Neuron 9:527-537). Indeed, a direct interaction of CSP with the α1A subunit of P/Q-type Ca2+ channels and an indirect interaction via G protein modulation was reported (Leveque et al., 1998, J. Biol. Chem. 273:13488-13492; Magga et al., 2000, Neuron 28:195-204). Drosophila CSP null mutants showed a wild-type like release of neurotransmitter when a Ca2+ ionophore was used to trigger exocytosis, implying that CSP functions in the coupling of the Ca2+ signal to secretion (Ranjan et al., 1998, J. Neurosci. 18:956-964). Consistent with this hypothesis, the temperature-sensitive inhibition of neuromuscular transmission in CSP null mutants is correlated with a rise in the intracellular Ca2+ concentration, suggesting that CSP increases the Ca2+ sensitivity of the exocytotic machinery (Dawson-Scully et al, 2000, J. Neurosci. 20:6039-6047). In Drosophila, an interaction between CSP and the t-SNARE syntaxin-1A has been reported (Nie et al., 1999, J. Neurosci. 19:10270-10279; Wu et al., 1999, Neuron 23:593-605) whereas in vertebrates, CSP was found to bind to synaptobrevin/VAMP but not to syntaxin (Leveque et al, 1998, J. Biol. Chem. 273:13488-13492).
Compelling evidence has been presented that CSP interacts with members of the heat-shock family Hsp70 (Braun et al., 1996, Neuropharmacology 34:1361-1369; Chamberlain and Burgoyne, 1997, Biochem. J. 322:853-858; Stahl et al., 1999, Eur. J. Cell Biol. 78:375-381). This interaction was originally found by means of an ATPase assay (Braun et al., 1996, Neuropharmacology 34:1361-1369). CSP strongly activated the Hsc70 ATPase. Thereafter, a direct binding of Hsc70 to the J domain of CSP could be shown (Stahl et al., 1999, Eur. J. Cell Biol. 78:375-381). Among other functions, members of the Hsp70 family, like Hsc70, act as molecular chaperones (Hendrick and Hartl, 1995, Faseb J. 9:1559-1569).
Molecular chaperone complexes act to catalyze the proper folding of newly synthesized proteins and renature proteins that have become denatured in response to various forms of cellular stress or injury (Hartl and Hayer-Hartl, 2002). Chaperones are also required for the translocation of proteins across cell membranes, play a role in macromolecular assembly and disassembly, mediate the degradation of proteins in the proteasome, and participate in various signal transduction pathways that induce gene transcription in response to stress, including those that act to suppress apoptosis.
Using the yeast two-hybrid system, a novel CSP binding partner, termed SGT, also has been identified. SGT was originally discovered because of its putative interaction with envelope proteins of two viruses, i.e. human immunodeficiency virus type 1 and parvovirus H-1 (Callahan et al., 1998, J. Virol. 72:5189-5197; Cziepluch et al., 1998, J. Virol. 72:4149-4156). A detailed sequence analysis revealed that SGT contains three tandem tetratricopeptide repeat domains (TPRs). The TPR domain is a degenerate 34 amino acid sequence found in a wide variety of proteins, present in tandem arrays of 3 to 16 motifs, which form scaffolds to mediate protein-protein interactions. Currently, more than 50 proteins are known to contain TPR motifs, present in organisms as diverse as bacteria and humans (Blatch and Lässle, 1999, Bioessays 21:932-939).
2. Role of Protein Folding in Neurodegenerative Diseases
Neurodegenerative disorders are increasingly common in Western societies. For example, in the United States alone, more than 6 million people are afflicted with Alzheimer's disease (AD) and Parkinson's disease (PD). AD is the most common senile dementia, affecting an approximately 10% of people over the age of 65, whereas PD is the most common movement disorder, affecting 1%-2% of this same population group. Other neurodegenerative diseases whose incidences have been rising include Huntington's disease (HD) and spinocerebellar ataxias (SCAs).
Although the genetic basis of most neurodegenerative diseases appears to be highly heterogeneous, many of these disorders display a common pattern of deposition of misfolded, aggregated, and ubiquitinated proteins followed by cell loss. For example, in AD, neurofibrillary tangles comprised of the Tau protein are found intracellularly and plaques comprised of β-amyloid (Aβ) peptides are found extracellularly in the neocortex and hippocampus where cell loss is most prominent (Selkoe, 2001, Physiol. Rev. 81:741-766). In PD, intracellular lesions known as Lewy bodies, comprised mainly of α-synuclein, are found in the cytoplasm of dopaminergic neurons of the substantia nigra, where cell loss subsequently occurs (Goedert, 2001, Nat. Rev. Neurosci. 2:492-501). In HD, the protein deposits are comprised of huntingtin, are form intranuclear and cytoplasmic inclusion bodies in neurons of the striatum and cerebral cortex (Zoghbi and Orr, 2000, Annu. Rev. Neurosci. 23:217-247). The presence of these aggregates suggests a defect in the ability of the cellular machinery that normally acts to prevent the formation or accumulation of misfolded proteins, namely protein chaperone complexes.
3. Current Treatments for Neurodegenerative Diseases and Their Limitations
Although the various neurodegenerative diseases are etiologically distinct, treatment strategies can be generally divided into two classes—palliative and “disease-altering.” Palliative strategies are those designed to alleviate symptoms and include, for example, the use of dopamine agonists or L-dopa in the treatment of PD to compensate for the deficits in dopamine neurotransmission caused by the loss of dopaminergic neurons from the substantia nigra, or the use of acetylcholinesterase inhibitors or cholinergic agonists in the treatment of AD to counteract the loss of cholinergic neurons from the neocortex and hippocampus. While somewhat beneficial in patient management, these approaches fail to alter the underlying pathophysiological process that lead to the diseases being treated
A variety of truly “disease-altering” approaches have been proposed or are being examined in preclinical and clinical studies of various neurodegenerative diseases. For example, pharmacological approaches designed to prevent the formation of the protein aggregates (e.g. β- or γ-secretase inhibitors to block the proteolytic processing of APP in AD), are being designed and tested. However, as many of the enzymes that lead to the formation of protein deposits are not involved in the processing of a wide variety of proteins, most of which are not involved in the disease process, the specificity and toxicity of these compounds is suspect.
Various immunological approaches are also being examined as a means of reducing the burden of protein plaques in the brain. For example, vaccination of AD patients with Aβ42 has been proposed as a means of clearing amyloid plaque from the brains of patients with AD. Unfortunately, at least some patients receiving this treatment in phase IIA clinical trials experienced significant and potentially life-threatening brain inflammation.
A great amount of attention has also been directed toward various restorative approaches to the treatment of neurodegenerative diseases, including transplantation or cell- or gene-based therapies. Though conceptually interesting, these approaches have many practical limitations. For transplantation, these include the lack of donor material for implantation, the failure of the transplanted material to engraft into the recipient and forge new synaptic connections, and the complexity and expense associated with brain surgery. In addition to these problems, cell-based therapies are further hindered by our paucity of knowledge concerning the identity of neuronal stem cells while gene-based therapies are beset by problems associated with gene delivery and gene regulation.
Although several of the “disease-altering” approaches detailed above show some promise, none are completely without inherent limitations. Thus, there is a strong need for the development of additional therapeutic modalities for the treatment of neurodegenerative diseases.
4. Utility of Molecular Chaperones in the Treatment of Neurodegenerative Diseases
Many publications have now demonstrated that overexpression of the Hsp70 and Hsp40 families of chaperones can suppress the aggregation and toxicity of polyglutamine-containing proteins such a huntingtin (Sherman and Goldberg, 2001). Perhaps the most compelling data in support of a role for chaperones in neurodegenerative diseases associated with protein misfolding comes from in vivo studies performed in fruit fly and mouse models of neurodegenerative disorders (Warrick et al., 1999; Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000; Cummings et al., 2001; Auluck et al., 2002). For example, overexpression of human Hsp70 completely suppressed the external eye defects mediated by the eye-specific expression of an expanded polyglutamine-containing protein in a Drosophila model of Machado-Joseph disease (Warrick et al., 1999). Overexpression of Hsp70 in a mouse model of spinocerebellar ataxia (SCA1) also reduced the neurodegeneration present in these animals, as well as ameliorating the behavioral phenotype of the treated animals (Cummings et al., 2001). Interestingly, in both studies, there appeared to be little or no change in the formation of polyglutamine-containing protein aggregates as determined by light microscopy (Warrick et al., 1999; Cummings et al., 2001). Similar results have been observed in Drosophila models of PD (Auluck et al., 2002). These findings suggest that chaperones act by changing the nature of the aggregates in such a way as to reduce their toxicity rather than completely preventing their formation. Alternatively, the beneficial effects of chaperone overexpression observed in these studies may stem from the other roles that chaperones play within the cell, such as protein trafficking or signal transduction. In either event, these results clearly show the value of chaperones as useful targets for pharmacological intervention in neurodegenerative diseases.
In accordance with the present invention, it has been discovered 1) cysteine string protein (CSP) forms a complex in the presynaptic nerve terminal with the heat shock protein Hsc70 and the small glutamine-rich tetratricopeptide (TPR) protein (SGT), 2) that this complex constitutes an ATP-dependent presynaptic chaperone machine that is essential in maintaining long-term synaptic function, and 3) that mice in which the CSP gene has been disrupted by insertional mutagenesis (CSP knockout mice) suffer from neuronal degeneration. These discoveries have permitted the development of assays and models that are useful in understanding the pathogenesis of various neurodegenerative diseases in which defects in protein folding have been implicated, in identifying additional endogenous or environmental factors that could contribute to the etiologies of these diseases, and in developing effective therapies for the prevention and/or treatment of these diseases.